Detection of BRAFV600E mutation of thyroid cancer in circulating tumor DNA by an electrochemical-enrichment assisted ARMS-qPCR assay

• An electrochemical-enrichment assisted amplification refractory mutation system-quantitative real-time PCR (EC-ARMS-qPCR) assay is introduced for detection of circulating tumor DNA (ctDNA). • The single nucleotide polymorphism (SNP) of BRAF V600E can be detected with high sensitivity. • EC-ARMS-qPCR assay is appropriate for detecting BRAF V600E in plasma of papillary thyroid cancer (PTC) patients. Qualitative and quantitative analysis of carcinogenic mutations in circulating tumor DNA (ctDNA) plays a unique role in the early screening and precise diagnosis of cancer. Herein, we introduced an electrochemical-enrichment assisted amplification refractory mutation system-quantitative real-time PCR (EC-ARMS-qPCR) assay to detect the BRAF V600E carcinogenic mutation of thyroid cancer in plasma samples. Cell-free DNA (cfDNA) were firstly enriched by an electric field-driven adsorption and desorption process, and then ctDNA in cfDNA were analyzed by a designed allele-specific qPCR assay. The EC-ARMS-qPCR assay provides good sensitivity and specificity for the identification of single nucleotide polymorphisms (SNPs) of BRAF V600E mutant ctDNA in a complex system, which can detect as low as 2 BRAF V600E mutant copies in Tris buffer and 25 BRAF V600E mutant copies in Tris buffer containing 50% plasma with a allele frequency of 0.01% (i.e., mutant-to-wild-type ratio of 1: 9999). The assay was assessed in plasma samples of patients with papillary thyroid cancer (PTC) and benign thyroid nodule (TN). The EC-ARMS-qPCR assay detected 40.74% and 10.00% BRAF V600E mutation ctDNA in plasma samples of 54 PTC patients and 20 TN patients, respectively. The overall concordance between BRAF V600E mutant ctDNA by EC-ARMS-qPCR assay and BRAF V600E mutant of matched pathological tissue genes by fine needle aspiration biopsy (FNAB) is 73.08%, suggesting that the EC-ARMS-qPCR assay is appropriate for detecting carcinogenic mutations in liquid biopsy samples.